The field of regenerative medicine offers the potential to significantly
impact a wide spectrum of healthcare issues, from diabetes to cardiovascular
disease. In particular, the design of tailored biomaterials, which possess
properties desired for their particular application, and the development
of superior implant environments, which seek to meet the nutritional needs
of the tissue, have yielded promising tissue engineering prototypes. In
this commentary, we examine the novel approaches researchers have made
in customized biomaterials and promoting angiogenesis that have led to
significant advancements in recent years.

The aim of this study was to understand the survival and differentiation
of neural stem/progenitor cells (NSPCs) cultured on chitosan matrices in
vivo in a complete transection model of spinal cord injury. NSPCs were
isolated from the subependyma of lateral ventricles of adult GFP transgenic
rat forebrains. The GFP-positive neurospheres were seeded onto the inner
lumen of chitosan tubes to generate multicellular sheets ex vivo. These
bioengineered neurosphere tubes were implanted into a completely transected
spinal cord and assessed after 5 weeks for survival and differentiation.
The in vivo study showed excellent survival of NSPCs, as well as differentiation
into astrocytes and oligodendrocytes. Importantly, host neurons were identified
in the tissue bridge that formed within the chitosan tubes and bridged
the transected cord stumps. The excellent in vivo survival of the NSPCs
coupled with their differentiation and maintenance of host neurons in the
regenerated tissue bridge demonstrates the promise of the chitosan tubes
for stem cell delivery and tissue regeneration.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease
lacking effective therapies. Cell replacement therapy has been suggested
as a promising therapeutic approach for multiple neurodegenerative diseases,
including motor neuron disease. We analyzed expanded mesenchymal stem cells
(MSCs) isolated from sporadic ALS patients and compared them with MSCs
isolated from healthy donors. MSCs were isolated from bone marrow by Percoll
gradient and maintained in culture in MSC Medium until the third passage.
Growth kinetics, immunophenotype, telomere length, and karyotype were evaluated
during in vitro expansion. Osteogenic, adipogenic, chondrogenic, and neurogenic
differentiation potential were also evaluated. No morphological differences
were observed in the MSCs isolated from donors or patients. The cellular
expansion potential of MSCs from donors and patients was slightly different.
After three passages, the MSCs isolated from donors reached a cumulative
population doubling higher than from patients but the difference was not
statistically significant. No significant differences between donors or
patients were observed in the immunophenotype analysis. No chromosomal
alteration or evidence of cellular senescence was observed in any samples.
Both donor and patient MSCs, after exposure to specific conditioning media,
differentiated into adipocytes, osteoblasts, chondrocytes, and neuron-like
cells. These results suggest that extensive in vitro expansion of patient
MSCs does not involve any functional modification of the cells, including
chromosomal alterations or cellular senescence. Hence, there is a good
chance that MSCs might be used as a cell-based therapy for ALS patients.

Department of Urology, Shinshu University School of Medicine, Matsumoto,
390-8621, Japan

This study is a preliminary investigation to determine if bone marrow-derived
cells, when implanted into freeze-injured urinary bladders, differentiate
into smooth muscle cells and reconstruct smooth muscle layers. Bone marrow
cells were harvested from femurs of male ICR mice and cultured in collagen-coated
dishes for 7 days. After 5 days of culture, the cells were transfected
with green fluorescent protein (GFP) genes for identification in recipient
tissues. Three days prior to implantation, the posterior urinary bladder
walls of female nude mice were injured with an iron bar refrigerated by
dry ice. Seven days after the culture and 3 days after the injury, adherent,
proliferating GFP-labeled bone marrow-derived cells (1.0 × 105
cells) were implanted into the injured regions. For controls, a cell-free
solution was injected. At 14 days after implantation, the experimental
urinary bladders were analyzed by histological, gene expression, and cystometric
investigations. Just prior to implantation, the injured regions did not
have any smooth muscle layers. After 14 days, the implanted cells surviving
in the recipient tissues were detected with GFP antibody. The implanted
regions had distinct smooth muscle layers composed of regenerated smooth
muscle marker-positive cells. The implanted GFP-labeled cells differentiated
into smooth muscle cells that formed into layers. The differentiated cells
contacted each other within the implanted region as well as smooth muscle
cells of the host. As a result, the reconstructed smooth muscle layers
were integrated into the host tissues. Control mice injected with cell-free
solution developed only few smooth muscle cells and no layers. Cystometric
investigations showed that mice with implanted the cells developed bladder
contractions similar to normal mice, whereas control mice did not. In summary,
mouse bone marrow-derived cells can reconstruct layered smooth muscle structures
in injured bladders to remediate urinary dysfunction.

Stem cell transplantation is emerging as a potential treatment option
for acute renal failure (ARF) because of its capability to regenerate tissues
and organs. To better understand the mechanism of cell therapy, in vivo
tracking cellular dynamics of the transplanted stem cells is needed. In
the present study, in vivo monitored magnetically labeled mesenchymal stem
cells (MSCs) were transplanted intravascularly into an ARF rat model using
a conventional magnetic resonance imaging (MRI) system. Rat bone marrow
MSCs were labeled with home synthesized Fe2O3-PLL,
and labeled (n = 6) or unlabeled MSCs (n = 6) were injected
into the renal arteries of the rats with ARF induced by the intramuscular
injection of glycerol. Using the same technique, labeled MSCs were also
injected into the rats assigned to a control group (n = 8). MR images
of kidneys were obtained before injection of MSCs as well as immediately,
1, 3, 5, and 8 days afterwards. MR findings were analyzed and compared
with histopathological and immunohistochemical results. These results showed
that the rat MSCs were successfully labeled with the home synthesized Fe2O3-PLL.
In both renal failure and intact rat models, the labeled MSCs demonstrated
a loss of signal intensity in the renal cortex on T2*-weighted MR images,
which was visible up to 8 days after transplantation. Histological analyses
showed that most of the labeled MSCs that tested positive for Prussian
blue staining were in glomerular capillaries, corresponding to the areas
where a loss in signal intensity was observed in the MRI. A similar signal
intensity decrease was not detected in the rats with unlabeled cells. These
data demonstrate that the magnetically labeled MSCs in the rat model of
ARF were successfully evaluated in vivo by a 1.5 T MRI system, showing
that the mechanisms of stem cell therapy have great potential for future
ARF treatment recipients.

Side population (SP) cells were isolated by FACS from a human amnion
mesenchymal cell (AMC) layer soon after enzyme treatment. The yield of
SP cells from AMC layer (AMC-SP cells) was about 0.1-0.2%. AMC-SP cells
grew well with cell doublings of 40-80 days of culture. FACS profiles and
immunocytostaining showed that AMC-SP cells were composed of two different
cells immunologically: HLA I-/II- and HLA I+/II-.
Oct-3/4 was detected in the nucleus of AMC-SP cells, when the culture was
examined at the third, sixth, and 10th passages. RT-PCR showed that AMC-SP
cells expressed the Oct-4, Sox-2, and Rex-1 genes.
Immunocytochemistry revealed that all AMC-SP cells were vimentin+,
CK19+, and nestin+. In addition, flow cytometry analysis
showed that SP cells had high expression of CD13, CD29, CD44, CD46, CD49b,
CD49c, CD49e, CD59, CD140a, and CD166 but low expression of CD 49d, and
CD51. No evidence of expression was obtained for CD34, CD45, CD49a, CD56,
CD90, CD105, CD106, CD117, CD133, CD271, or Flk-1. Upon appropriate differentiation
protocols, AMC-SP cells differentiated to several cell lineages such as
neuroectodermal, osteogenic, chondrogenic, and adipogenic cells. These
results indicate that AMC-SP cells have multilineage potential to several
cell lineages with unique immunological characteristics such as HLA I-/II-
or HLA I+/II-. AMC-SP cells should be of considerable
value for regenerative medicine because they do not induce acute rejection
after allotransplantation, they do not cause ethical issues, and there
is no limit of supply.

The stromal stem cell fraction of many tissues and organs has demonstrated
to exhibit stem cell properties such as the capability of self-renewal
and multipotency, allowing for multilineage differentiation. In this study,
we characterize a population of stromal stem cells derived from menstrual
blood (MenSCs). We demonstrate that MenSCs are easily expandable to clinical
relevance and express multipotent markers such as Oct-4, SSEA-4, and c-kit
at the molecular and cellular level. Moreover, we demonstrate the multipotency
of MenSCs by directionally differentiating MenSCs into chondrogenic, adipogenic,
osteogenic, neurogenic, and cardiogenic cell lineages. These studies demonstrate
the plasticity of MenSCs for potential research in regenerative medicine.

Directed endodermal differentiation of murine embryonic stem (ES) cells
gives rise to a subset of cells with a hepatic phenotype. Such ES cell-derived
hepatic progenitor cells (ES-HPC) can acquire features of hepatocytes in
vitro, but fail to form substantial hepatocyte clusters in vivo. In this
study, we investigated whether this is due to inefficient engraftment or
an immature phenotype of ES-HPC. ES cells engrafted into recipient livers
of NOD/SCID mice with a similar efficacy as adult hepatocytes after 28
days. Because transplanted unpurified ES-HPC formed teratomas in the spleen
and liver, we applied an albumin promoter/enhancer-driven reporter system
to purify ES-HPC by cell sorting. RT-PCR analyses for hepatocyte-specific
genes showed that the cells exhibited a hepatic phenotype, lacking the
expression of the pluripotency marker Oct4, comparable to cells of day
11.5 embryos. Sorted ES-HPC derived from b-galactosidase
transgenic ES cells were injected into fumaryl-acetoacetate-deficient (FAH-/-)
SCID mice and analyzed after 8 to 12 weeks. Staining with X-gal solution
revealed the presence of engrafted cells throughout the liver. However,
immunostaining for the FAH protein indicated hepatocyte formation at a
very low frequency, without evidence for large hepatocyte cluster formation.
In conclusion, the limited repopulation capacity of ES-HPC is not caused
by a failure of primary engraftment, but may be due to an immature hepatic
phenotype of the transplanted ES-HPC.

Possible myogenic differentiation of SSEA-1- and OCT-4-positive murine
embryonic stem cells (ESCs) and embryoid bodies (EBs) was studied in vitro
and in vivo. In vitro, ESC- or EB-derived ESCs (EBs/ESCs) showed only traces
of Pax 3 and 7 expression by immunocytochemistry and Pax 3 expression by
immunoblot. By RT-PCR, myogenic determinant molecules (myf5, myoD, and
myogenin) were expressed by EBs/ESCs but not by ESCs. However, in such
cultures, very rare contracting myotubes were still present. Suspensions
of LacZ-labeled ESCs or EBs were injected into anterior tibialis muscles
(ATM) of different cohorts of mice for the study of their survival and
possible myogenic differentiation. The different cohorts of mice included
isogenic adult 129/Sv, nonisogenic CD1 and mdx, as well as mdx immunosuppressed
with 2.5 mg/kg daily injections of tacrolimus. Ten to 90 days postinjections,
the injected ATM of nonisogenic mice did not contain cells positive for
LacZ, SSEA-1, OCT-4, or embryonic myosin heavy chain. The ATM of intact
mdx mice contained very rare examples of muscle fibers positive for dystrophin
and/or embryonic myosin heavy chain. In the ATM of the isogenic normal
and the immunosuppressed mdx mice, as expected, large teratomas developed
containing the usual diverse cell types. In some teratomas of immunosuppressed
mdx mice, small pockets of muscle fibers expressed dystrophin and myosin
heavy chain. Our studies indicated that in muscles of animals nonisogenic
with the used ESCs, only very rare ESCs survived with myogenic differentiation.
These studies also indicated that ESCs will not undergo significant, selective,
and preferential myogenic differentiation in vitro or in vivo in any of
the models studied. It is probable that this strain of murine ESC requires
some experimentally induced alteration of its gene expression profile to
secure significant myogenicity and suppress tumorogenicity.

Duchenne muscular dystrophy is a recessive disease due to a mutation
in the dystrophin gene. Myoblast transplantation permits to introduce the
dystrophin gene in dystrophic muscle fibers. However, the success of this
approach is reduced by the short duration of the regeneration following
the transplantation, which reduces the number of hybrid fibers. Our aim
was to verify whether the success of the myoblast transplantation is enhanced
by blocking the myostatin signal with an antagonist, follistatin. Three
different approaches were studied to overexpress follistatin in the muscles
of mdx mice transplanted with myoblasts. First, transgenic follistatin/mdx
mice were generated; second, a follistatin plasmid was electroporated in
mdx
muscles, and finally, follistatin was induced in mdx mice muscles
by a treatment with a histone deacetylase inhibitor. The three approaches
improved the success of the myoblast transplantation. Moreover, fiber hypertrophy
was also observed in all muscles, demonstrating that myostatin inhibition
by follistatin is a good method to improve myoblast transplantation and
muscle function. Myostatin inhibition by follistatin in combination with
myoblast transplantation is thus a promising novel therapeutic approach
for the treatment of muscle wasting in diseases such as Duchenne muscular
dystrophy.

Restoration of cutaneous pigmentation has been achieved in stable vitiligo
by autologous melanocyte transplantation. This study was aimed to develop
a methodology to deliver melanocytes to vitiliginous area following their
processing and culture in a centralized facility. Here we report a methodology
to culture melanocytes on carrier films, transport the cells, and graft
them on vitiliginous areas. The salient features of this study include:
1) development of polylactic acid (PLA) films that support melanocyte attachment,
growth, and delivery; 2) establish transport conditions for skin biopsies
from hospitals; 3) establish transport conditions for cultured cells from
cell processing center to hospitals. Results suggest that PLA films could
serve as carriers for melanocytes during transport. "pside-down"application
of the graft results in the migration of cells from the films into the
dermabraded area. The transport conditions ensure cell viability for 96
h. This system could help clinicians, who do not have access to cell culture
facilities, transplant cultured melanocytes in a cost-effective manner.